1. Field of the Invention
The present invention relates generally to the fields of optics and microscopy. More particularly, it concerns apparatus and methods for analyzing samples using fiber-optic confocal imaging techniques.
2. Description of Related Art
Currently, two methods are available for imaging at the cellular level in the body: Optical Coherence Tomography (OCT) (Izatt et al., 1994; Huang et al., 1991; Schmitt et al., 1993; Yadlowsky et al., 1995; Swanson et al., 1993; Schmitt et al., 1994) and confocal imaging (Petroll et al., 1993; Jester et al., 1991; Rajadhyaksha et al., 1995a; Gmitro and Aziz, 1993; Massig et al., 1994; Masters and Thaer, 1994; Giniunas et al., 1993a; 1993b; Delaney et al., 1994).
OCT uses interference techniques with low-coherence light sources to select the light coming from a distinct depth (Huang et al., 1991). The basic system uses a Michaelson interferometer with the tissue sample in one arm and a reference mirror in another arm. When the reflections from both arms are combined at the detector, an interference maximum or minimum is detected when the reflections from both arms are matched in optical path length (time-of-flight). The strength of the interference is proportional to the amount of light reflected from the corresponding optical path length within the tissue. The temporal frequency of the interference maximum and minimum can be modulated by translating the reference mirror at a constant velocity or by stretching the path length in the reference arm with a piezo-electric transducer at the modulation frequency. These systems use heterodyne detection of the modulated interference signal to detect as little as 5xc3x9710xe2x88x9210 of the incident light (Huang et al., 1991). The axial resolution of OCT depends on the coherence length of the illumination source, which is in the range of 10 to 20 xcexcm (Swanson et al., 1993) for semi-conductor sources. More recent work with mode-locked Ti:Sapphire and Forrestrite lasers have yielded coherence lengths as small as 1.8 xcexcm (SPIE Proceedings, 1997). The lateral resolution is determined by the diffraction-limited spot size in the tissue.
OCT forms a cross-sectional image of the tissue by mapping the intensity of the reflected light as the sampling point is translated in the axial and lateral dimensions. The sampling point is moved in depth by the translation of the reference mirror. The lateral dimension is achieved by translation of the optics over the surface of the tissue. Much of the in vivo imaging done with OCT has been in the eye (Swanson et al., 1993). Some researchers have also attempted to use the technique to image scattering tissue such as human skin, however, have not obtained images of individual cells due to the lack of spatial resolution (Yadlowsky et al., 1995). Thus, while OCT has a high sensitivity, it has not demonstrated the spatial resolution necessary to image cellular structure. Although the new mode-locked laser gives OCT the potential to achieve the desired resolution, the cost and complexity of these lasers make them impractical for clinical use.
The first attempts at in vivo confocal imaging have been done with a modified scanning Nipow disk microscope (Petroll et al., 1993; Jester et al., 1991). A Nipow disk refers to fiat disk which has a staggered array of pinhole apertures spread over the entire disk. At one instant in time, one of the apertures passes the illumination light and detects the reflected confocal light. As the disk spins, the aperture being used for illumination and detection moves around the disk, thereby, imaging the entire sample. The disks can spin at very high speeds to produce images at video rates. These systems have been used to image in vivo cornea and several organs of a rat which had been exposed by laparotomy. The best spatial resolution reported to date is approximately 7 xcexcm, with no mention of the sensitivity or corresponding maximum penetration depth. One limitation of this apparatus is the fact that these microscopes are susceptible to misalignment of the disk.
More recently, a confocal system has been developed with a spatial resolution sufficient to image individual skin cells in a living human (Rajadhyaksha et al., 1995b). The instrument is a simplified confocal microscope with a standard pinhole aperture and scanning mirrors. A high numerical apertune (NA) oil-immersion objective lens is used in contact with the skin to achieve a lateral resolution of approximately 2 xcexcm. No values have been reported for the sensitivity of the system, however it is capable of imaging the entire thickness of the forearm epithelium and into the rete ridges of the underlying stroma using 830 nm light. Images of cell size and nuclear to cytoplasmic ratio obtained with this system agree well with those measured from biopsies, validating the concept that in vivo confocal imaging can be used to assess tissue morphology. However, the size and configuration of illumination optics prevents use of this system to image tissues within moderately accessible cavities such as the cervix or mouth.
Several authors have proposed fiber optic systems for in vivo confocal imaging (Massig et al., 1994; Masters and Thaer, 1994; Giniunas et al., 1993a; Giniunas et al., 1993b; Delaney et al., 1994) based upon fiber optics. These designs implement confocal detection through a single fiber optic. These designs also incorporate some method of translating the endpiece optics in the axial and transverse directions to form an image. Designing an endoscopic system encompassing a miniature, high speed mechanical scanning system with high spatial resolution is difficult.
Another approach to a fiber optic design (Gmitro and Aziz, 1993) uses a fiber optic imaging bundle as a confocal image conduit between the endpiece optics and a confocal microscope. The function of the confocal microscope was to scan the illumination spot across the fiber bundle and to detect the emerging light. This arrangement does avoid the need for a mechanical translation system in the endpiece, however a commercial confocal microscope is expensive and cumbersome, limiting its usefulness as a practical clinical tool. In addition, the high absorption and scattering of the tissue will not allow the fluorescence excitation and emission light to penetrate the entire depth of the epithelium.
More recently, the same design has been implemented for reflection imaging using white light (Juskaitis et al., 1997) Reflection imaging is capable of penetrating to greater depths; however, the use of a white light source will limit the illumination power available to the system. A consequence of the limited illumination power will be a limited penetration depth due to loss of signal in scattering tissue. The rationale given for using white light is eliminating the speckle observed when imaging a resolution test target with laser light.
Although the potential of both in vivo confocal imaging with subcellular resolution and of fiber optic confocal imaging has been demonstrated, there is currently not a system which provides the imaging capabilities required for imaging tissues in vivo within the physical constraints necessary to achieve sufficient resolution and magnification of tissues within a living organism.
The system described by Juskaitis et al. (1997) uses a combination of angle polishing and index matching with glycerin to xe2x80x9cpreventxe2x80x9d specular reflection from the faces of the fiber bundle. Because the fiber bundle requires a difference in index between the individual fiber cores and cladding to function, it is not possible to eliminate or xe2x80x9cpreventxe2x80x9d the reflection from the fiber face at the proximal end (i.e. the end that light is injected into). Instead, the reflections can be minimized by using a matching oil with an index half way between the index of the cores and the cladding. Indeed, Juskaitis et al. use such an oil. While it is theoretically possible to eliminate the reflections from the distal end by using an oil with an index exactly equal to the index of the fiber cores, it is not practically feasible due to manufacturing limitations and variations.
If the laser has a coherence length greater than the separation between either fiber face and the image plane, the reflections from the fiber faces will interfere with the light reflected from the image plane. The argon laser used by Juskaitis et al. has a coherent length of several meters. It is likely that the length of fiber bundle used was less than two meters. Thus, the unstable speckle pattern they report is a result of interference between one, or both, of the reflections from the fiber faces and the light from the image plane. The selection criteria of the laser of the present invention is a coherence length less than the separation between the distal end of the fiber and the image plane to avoid this problem.
A scanned optical fiber confocal microscope is described by Deckensheets and Kino (1994). However, the fresnel objective lens used in this microscope was not able to achieve the high NA needed for imaging cellular structure.
U.S. Pat. No. 5,659,642, describes a confocal microscope and endoscope similar to that of the present invention. However, unlike the present invention, this microscope lacks a method for controlling the specular reflections from the faces of the fiber bundle.
The present invention overcomes these and other limitations in the prior art by providing apparatus comprising a confocal fiber optic imaging system. In one embodiment, the device has been shown to achieve the resolution and imaging necessary in vivo to permit the detection and diagnosis of precancerous lesions in tissues such as those involving the epithelium. The apparatus disclosed provide clinical tools which dramatically improve recognition and monitoring of biological specimens such as epithelial pre-cancers of the oral mucosa, uterine cervix, urinary bladder, colon, as well as other organs with high incidence of epithelial cancer. The apparatus has produced images of physiological structure with micron resolution at 15 frames per second. Moreover, a series of lenses designed and constructed to incorporate the fiber optic bundle directly into the imaging system was developed to overcome the specular reflections from the face of the fiber optic bundle.
In one sense, the invention encompasses the design and development of a fiber optic confocal imaging system which uses reflected light to produce images of tissue with several micron resolution. This system provides the user with images of the cellular structure and organization of the sampled tissue. This information can be used to determine the morphology of tissue and its potential for diseases such as cancer.
A confocal optical system places an aperture in a conjugate image plane to reject any light which is not reflected from the focus of the optical system (Inoue, 1995). The confocal technique of selecting only the light reflected from the focal plane is sometimes referred to as optical sectioning.
The present invention provides a fiber optic confocal microscope which comprises a plurality of optical fibers packed side by side in a bundle to form a characteristic image of the sample at the focal plane from the reflected illumination light in real time. The apparatus utilizes index matching to detect the sample reflection of biological sample.
The apparatus may quantitatively analyze a variety of samples, including biological samples. A confocal reflectometer according to the present disclosure may measure the reflected light from a single point. A reflectometer built in accordance with the present disclosure has demonstrated resolution near the diffraction limit and the sensitivity to detect a 0.05 refractive index mismatch under 3 optical depths of scattering.
Lenses to incorporate a fiber bundle into a confocal microscope were designed and assembled. An apparatus according to the present disclosure has shown that specular reflection from fiber optic bundle faces may be controlled, and lateral resolution in the range of approximately 5 xcexcm may be achieved. Likewise, an axial resolution of approximately 15 xcexcm has been obtained using the disclosed apparatus.
In one aspect, the invention is a confocal imaging apparatus for analyzing a sample including a radiation source, a scan system, a scan lens, a plurality of fibers, a distal index matching agent, a coupling lens, and a detector. The radiation source is configured to emit incident radiation. The scan system is in optical communication with the radiation source and is configured to controllably deflect the incident radiation. The scan lens is in optical communication with the scan system and is configured to focus the incident radiation. The plurality of fibers have a proximate end and a distal end. The proximate end is in optical communication with the scan lens and is configured to receive the incident radiation focused from the scan lens. The distal index matching agent is coupled to the distal end and is configured to reduce specular reflection from the plurality of fibers. The coupling lens is in optical communication with the distal end and is configured to focus the incident radiation toward the sample to produce secondary radiation from the sample. The detector is in optical communication with the scan system and is configured to receive at least a portion of the secondary radiation and to produce a signal corresponding therewith.
In other aspects, the incident radiation may include near infrared radiation. The radiation source may be a Ti:Sapphire laser. The radiation source may be a diode pumped Nd:YAG laser. The scan system may include a pair of orthogonal galvanometers. The scan system may include a spinning polygon. The scan system and the scan lens may be adapted to illuminate a single fiber of the plurality of fibers. The apparatus may also include a proximal polarizing agent in operative relation to the proximal end and configured to reduce specular reflection from the plurality of fibers. The apparatus may also include a depth translation system in operative relation with the plurality of fibers. The depth translation may include a translation stage. The depth translation system may include a suction agent. The suction agent may include a tube having a plurality of channels, and at least one of the channels may be adapted to deliver saline while at least another one of the channels may be adapted for suction. The centers of the plurality of fibers may be separated by about 5 microns. At least one of the plurality of fibers may include a core and a cladding, and the distal index matching agent may include a fluid having an index of refraction substantially equal to an index of refraction of the core. The apparatus may also include a beam splitter in optical communication with the radiation source and the detector. The, beam splitter may include a wedge angle. The scan system may be configured to controllably deflect the incident radiation in a raster pattern. The apparatus may also include an aperture positioned between the coupling lens and the detector. One of the plurality of fibers may be an illuminated fiber transporting the secondary radiation toward the detector, and the aperture may have a diameter adapted to block at least a portion of the secondary radiation emanating from a proximate end of one or more fibers adjacent the illuminated fiber. The apparatus may also include a controller coupled to the scan system and to the detector. The apparatus may also include control electronics and a video card coupled to the controller. The control electronics may be adapted to provide one or more timing signals to the video card. The apparatus may also include an objective in optical communication with the coupling lens, and a magnification of the coupling lens may be adapted to fill the objective with the incident radiation. The apparatus may have a lateral resolution of about 5 microns.
In another respect, the invention is a confocal imaging apparatus for analyzing a sample and includes a laser, a scan system, a scan lens, a plurality of fibers, a proximal index matching agent, a distal index matching agent, a coupling lens, and a detector. The laser is configured to emit incident radiation. The scan system is in optical communication with the laser and is configured to controllably deflect the incident radiation in a raster pattern. The scan lens is in optical communication with the scan system and is configured to focus the incident radiation in the raster pattern. The plurality of fibers have a proximate end and a distal end. The proximate end is in optical communication with the scan lens and is configured to receive the incident radiation focused from the scan lens in the raster pattern. The proximal index matching agent is coupled to the proximate end and is configured to reduce specular reflection from the plurality of fibers. The distal index matching agent is coupled to the distal end and is configured to reduce specular reflection from the plurality of fibers. The coupling lens is in optical communication with the distal end and is configured to focus the incident radiation in the raster pattern toward the sample to produce secondary radiation from the sample. The detector is in optical communication with the scan system and is configured to receive at least a portion of the secondary radiation and to produce a signal corresponding therewith.
In other aspects, at least one of the plurality of fibers may include a core and a cladding, and the distal index matching agent may include a fluid having an index of refraction substantially equal to an index of refraction of the core. At least one of the plurality of fibers may include a core and a cladding, and the proximal index matching agent may include a fluid having an index of refraction between an index of refraction of the core and an index of refraction of the cladding. The fluid may have an index of refraction of about halfway between the index of refraction of the core and the index of refraction of the cladding. The apparatus may also include a depth translation system in operative relation with the plurality of fibers. The depth translation system may include a translation stage. The depth translation system may include a suction agent. The apparatus may also include a controller coupled to the scan system and to the detector.
In another respect, the invention is an endoscopic confocal imaging apparatus for in vivo analysis of a sample, including a confocal system and an endoscope. The confocal system includes a radiation source, a scan system, a scan lens, a plurality of fibers, and a detector. The radiation source is configured to emit incident radiation. The scan system is in optical communication with the laser and is configured to controllably deflect the incident radiation. The scan lens is in optical communication with the scan system and is configured to focus the incident radiation. The plurality of fibers have a proximate end and a distal end. The proximate end is in optical communication with the scan lens and is configured to receive the incident radiation focused from the scan lens. The detector is in optical communication with the scan system. The endoscope includes a distal index matching fluid reservoir, a coupling lens, and an endoscopic tube. The distal index matching fluid reservoir is configured to sealably contain a distal index matching fluid. The reservoir is coupled to the distal end, and the fluid is configured to reduce specular reflection from the plurality of fibers. The coupling lens is in optical communication with the distal end and is configured to focus the incident radiation toward the sample to produce secondary radiation from the sample detectable by the detector to produce a signal corresponding therewith. The endoscopic tube is configured to house the distal end, the reservoir, and the coupling lens.
In other aspects, the apparatus may also include an objective in optical communication with the coupling lens. A magnification of the coupling lens may be adapted to fill the objective with the incident radiation. The apparatus may also include a fiber shield configured to house and protect at least a portion of the plurality of fibers. The scan system may be configured to controllably deflect the incident radiation in a raster pattern. The apparatus may also include a suction hood coupled to the endoscope. The apparatus may also include a controller coupled to the scan system and to the detector.
In another respect, the invention is a method for confocal imaging of a sample. Incident radiation is emitted from a radiation source. Incident radiation is controllably deflected with a scan system in optical communication with the radiation source. Incident radiation is focused with a scan lens in optical communication with the scan system. The incident radiation focused from the scan lens is received with a proximate end of a plurality of fibers, the proximate end being in optical communication with the scan lens. Specular reflection from the plurality of fibers is reduced with a distal index matching agent coupled to a distal end of the plurality of fibers. The incident radiation is focused toward the sample to produce secondary radiation from the sample with a coupling lens in optical communication with the distal end. The secondary radiation focused from the coupling lens is received with the distal end. The secondary radiation is focused through the scan system with the scan lens. At least a portion of the secondary radiation is detected with a detector in optical communication with the scan system. A signal corresponding to the secondary radiation detected by the detector is produced to image the sample.
In other aspects, the coupling lens, the distal end, and the distal index matching agent may make up an endoscope, and the imaging of the sample may include in vivo endoscopic imaging of the sample. The controllably deflecting of the incident radiation may include deflecting the incident radiation in a raster pattern. The method may also include reducing specular reflection from the plurality of fibers with a proximal index matching agent coupled to a proximal end of the plurality of fibers. The sample may include biological tissue. The sample may include an integrated circuit wafer, or portion thereof. The method may also include enhancing contrast of the sample with a contrast agent. The contrast agent may include 5-aminolevulinic acid. The contrast agent may include acetic acid. The acetic acid may consist of about 6% acetic acid. The method may also include modifying a focus depth with a depth translation system in operative relation with the plurality of fibers. The depth translation system may include a suction agent configured to displace at least a portion of the sample by suction. The depth translation system may include a translation stage. The imaging may include cross sectional imaging.